Polysaccharides are complex carbohydrates comprised of polymers made up of many monosaccharides joined together by glycosidic linkages. Polysaccharides include storage polysaccharides such as starches and glycogen, and structural polysaccharides such as cellulose, acid polysaccharides containing sialic acid or sulfuric ester groups, and chitin. Polysaccharides have diverse biological functions including long term storage of sugars (for energy), and structural support and protection to organisms; cellulose is a major component of cell walls in plants and bacteria, and chitin is a component of fungi cell walls and exoskeletons of arthropods. Polysaccharides are also involved in molecular recognition (antigens) by cells of the immune system.
Glycogen is a storage polysaccharide comprised of glucose. Most of the glucose units are linked by α-1,4 glycosidic bonds, but approximately 1 in 12 glucose residues also makes α-1,6 glycosidic bond with a second glucose, which results in the creation of a branch. Glycogen is present in a wide variety of tissues, including skin, liver, parathyroid glands and skeletal and cardiac muscle. Detection of glycogen is used in clinical settings for the diagnosis of diseases including cancer, infections (fungus, Chlamydia), diabetes (Type 1), myopathies, glycogen storage disease (Pompe disease), and other glycogenoses. Histochemical detection of glycogen also is used to identify structures such as connective tissues, mucus, and basal laminae, corporal amylacea, polygucosan bodies and other substances in biological samples, all of which contain a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans). Specifically, PAS is used to stain neutral mucopolysaccharides, such as those in glands of the GI tract and in prostate; simple acidic polysaccharides containing sialic acid, such as those found in epithelial cells; and complex sulfated acid polysaccharides such as those found in adenocarcinomas.
Historically, imaging of polysaccharide, e.g., glycogen, content in cells and tissues has been measured qualitatively using the Periodic acid-Schiff (PAS) reaction. PAS stains glycogen and other polysaccharide molecules based on periodic acid-induced oxidative cleavage of carbon-to-carbon bonds in 1,2 glycols to form dialdehydes that react with fuchsin-sulphurous acid in the Schiff's reagent (pararosanilin and sodium metabisulfate) to yield a magenta-like stain (Bancroft and Stevens, Theory and practice of histological techniques. Churchill Livingstone, Edinburgh, p. 436, 1977). More recently, the PAS technique has been combined with optical density measurements, and compared to optical densities of external standards with known concentrations of glycogen, to convert to quantitative glycogen values (Schaart et al., Histochem Cell Biol. 2004; 122:161-169).
Fluorescence intensity measurement is another approach used commonly to quantify relative or absolute amounts of select biological materials. Fluorescence occurs where a molecule in an excited state (i.e., excited by absorption of EM radiation) emits light as it falls back to the lower energy state. The emission typically is at a longer wavelength than the wavelength of the excitatory radiation absorbed, and is in the visible range of the electromagnetic spectrum. Quantitation of fluorescence intensity is straightforward, can be readily achieved with instruments ranging from microtiter plate readers, fluorescence scanners, fluorescence and confocal microscopes. The microtiter plate reader or fluorescence scanner format is particularly suitable for simultaneous quantification and comparison of a large number of samples, such as is necessary in high-throughput applications.
Near-infrared fluorescence is a technique useful for in vivo imaging, e.g., for detecting tumors. This method is based on the fact that living tissue transmits fluorescence with wavelengths in the near-IR (about 700 nm) more efficiently than it transmits light with wavelengths in the visible range, due to increased photon penetration. Both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules for imaging. However, this technique is for in vivo detection and imaging and has not been employed to quantitate material ex vivo in biological samples.
Autofluorescence of PAS stain in the visible red range (excitation ˜540-580 nm; emission ˜600-640 nm) has been described (Changaris et al., Histochemistry. 1977; 52:1-15; Schaart et al., supra) and used for quantitative measurement of glycogen in liver sections (Changaris et al., supra). Although useful, fluorescence measurements in the visible range are subject to high background due to auto-fluorescence of biological materials in the visible range, and poor detection of signal penetration from thick tissue sections.
One strategy used to overcome the limitations of the visible range fluorescence measurements is fluorescence imaging microscopy (Brenner et al., J Histochem Cytochem. 1976. 24:100-11). This method requires the assembly and integration of sophisticated equipment including a light source and appropriate filters for excitation, a microscope with sensitive optics, fine focus, an XY stage control for spatially-resolved sample imaging, emission filters, a sensitive camera for image capture, and a computer for microscope control, equipped with image collection and processing software for documentation. Focusing, XY positioning, filter configuration, image capture and collection are carried out independently for each sample and fluorophore. Image processing software is used to merge the two images from each fluorophore of a given sample to view the spatial localization of the two fluorophores simultaneously. This final merged image is a qualitative image of the spatial distribution of the fluorophore probes. Additional image quantification software, that is designed to select a region of interest and quantify fluorescence intensity within that region for each fluorophore is used to quantify the fluorescence. Thus, fluorescence is measured within areas of cells or tissues of interest, and in a region defined as background. Each fluorescence measurement is corrected for the size of the region, and then background subtracted. The exquisite spatial and focal resolution that is achieved with this methodology allows for sufficient signal to background detection in the visible range. However, this method is tedious as each sample is imaged one at a time, and the captured image is processed and quantitated one at time. As the number of samples increase, and the number of fluorophores that are imaged per sample increase, data acquisition and processing time is proportionally increased, and throughput is proportionally decreased.
Accordingly, there remains a need in the art for an improved method to quantify polysaccharides, particularly glycogen, in biological samples.